Many agriculturally important crops are subject to infection by plant viruses and viroids. These viruses can seriously damage a crop and drastically reduce its economic value to the grower. This eventually leads to a higher cost of the goods to the ultimate consumer. Attempts to control or prevent infection of a crop by a plant virus have been made, but until recently none have been completely satisfactory. Accurate global figures for crop losses due to viruses alone are not available. However, some idea of the scale can be obtained by considering that plant disease losses worldwide were estimated to be in excess of $60 billion per year in 1986.
Human and animal diseases associated with viral infections present a continuing challenge for development of new products, treatments and therapies. A fresh approach in the treatment of infectious diseases arose from demonstration of the ability to successfully introduce foreign genes into eukaryotic cells. Gene therapy, which is the introduction of genetic material into mammalian somatic cells in order to treat malignant, infectious or inborn genetic diseases, is considered to be a realistic and desired method of treatment for viral-induced infections, including malignancies.
One of the most important goals today is the development of virus resistant plants and animals. Current methods for viral protection involve the use of a virus component to stimulate an antiviral state in the host. Such protection appears not to be broad, but specific to related species. Plants or animals exhibiting broad spectrum viral resistance are not available.
The theory of virus-derived resistance proposes that pathogen resistance genes can be derived from a pathogen's own genetic material. Numerous examples of virus-resistance have been reported for many different plant RNA viruses in a wide range of plant species. Most examples of virus-resistance involve transgenic plants engineered to express a viral coat protein (CP) or a segment of a replicase gene (for reviews, see Beachy et al., Ann. Rev. Phytopathol. 28:451-474; Wilson, 1993, Proc. Natl. Acad. Sci. 90:3134-3141). As a general rule, transgenic plants accumulating one of these viral proteins are often resistant to that particular virus and closely related viruses. For example, the coat protein (CP) of the protectant virus was thought to be primarily responsible, either by preventing particle disassembly or by re-encapsidating the incoming genome of the more severe challenge virus. However, viroids [240- to 380-nucleotides-long, naked circular single-stranded RNA (ssRNA) pathogens] and mutant viruses making assembly-defective or no detectable CP could also cross-protect against their more severe relatives. Although many examples of virus resistance have been documented, in general, the mechanism(s) underlying resistance remains to be clearly defined.
Although plant transformations with viral coat protein sequences, partial replicase gene sequences, antisense RNA sequences to key viral genes, and satellite viruses have resulted in moderate levels of disease resistance highly specific to only certain host-virus interactions, problems related to recombination of viral genes inserted into hosts have increased the biohazard potential of these approaches. To our knowledge, there is no viable strategy to develop viroid resistant plants.
There exists, therefore, a continuing need for an improved method and effective means for controlling virus- and viroid-infection in plants and animals, and for providing transgenic species that are broadly resistant to multiple viruses and viroids.
The interferon system is the primary host defense against viral infection in vertebrates [Lengyel (1982) Ann. Rev. Biochem. 51:251-282]. It is thought that dsRNA, which is produced during most viral infections, can act as an inducer for interferon synthesis [Field et al. (1967) Proc. Natl. Acad. Sci 58:1004-1010]. Once synthesized, interferons are secreted, bind to specific receptors on cells, and induce an antiviral state. Cells in the antiviral state are more resistant to infection by most viruses. Several interferon-induced enzymes are involved in the establishment of the antiviral state. One of the interferon-induced enzymes is the P.sub.1 /elF-2.alpha. protein kinase that can autophosphorylate and phosphorylate exogenous substrates including the eukaryotic protein synthesis initiation factor elF-2, and histone proteins [Samuel et al. (1986) Meth. Enzymol. 119:499-516; Jacobs and Imani (1988) J. Interferon Res. 8:821-830]. Autophosphorylation and activation of the P.sub.1 /elF-2.alpha. kinase requires binding to dsRNA [Galabru and Hovanessian (1987) J. Biol. Chem. 262:15538-15544]. Once activated, the P.sub.1 /elF-2.alpha. kinase can phosphorylate elF-2 on its a subunit and alter the interaction of elF-2 with the GDP/GTP exchange factor elF-2B, leading to an inhibition of protein synthesis [Safer (1983) Cell 33:7-8].
A number of viruses, including adenovirus, influenza virus, human immunodeficiency virus type 1, reovirus, Epstein-Barr virus, and vaccinia virus have been reported to produce or induce inhibitors of the P.sub.1 /elF-2.alpha. kinase [Kitajewski et al. (1986) Mol. Cell. Biol. 6:4493-4498; Lee et al. (1990) Proc. Natl. Acad. Sci. 87:6208-6212; Gunnery et al. (1990) Proc. Natl. Acad. Sci. 87:8687-8691; Imani and Jacobs (1988) Proc. Natl. Acad. Sci 85:7887-7891; Clarke et al. (1990) Eur. J. Biochem. 193:635-641; Whitaker-Dowling and Younger (1983) Virology 131:128-136; Paez and Esteban (1984) Virology 134:12-28; and Rice and Kerr (1984) J. Virol. 50:229-236] and consequently evade one of the antiviral modes of interferons. For example, large quantities of VAI RNA are produced in adenovirus-infected cells. The VAI RNA can bind to the P.sub.1 /elF-2.alpha. kinase and prevent its activation [Galabru et al (1989) Eur. J. Biochem. 178:581-589]. For reovirus, a dsRNA binding protein, .sigma.3, functions as the P.sub.1 /elF-2.alpha. kinase inhibitor by binding to and competing for activator dsRNA (Imani and Jacobs 1988, supra). In the case of influenza virus, the kinase inhibitor is a cellular protein activated during viral infection (Lee et al. 1990, supra). Adenovirus and certain strains of reovirus have been demonstrated to be resistant to the antiviral effects of interferon [Kitajewski et al. (1986) supra; Jacobs and Ferguson (1991) J. Virol 65:5102-5104]. For adenovirus, deletion of the gene for VAI RNA leaves the virus interferon sensitive (Kitajewski et al. 1986, supra).
Replication of vaccinia virus in mouse L cells is resistant to interferon treatment [Younger et al. (1972) J. Virol. 10:171-180]. Vaccinia virus infection can also rescue other viruses, such as vesicular stomatitis virus (VSV) and encephalomycarditis virus (EMCV) (Whitaker-Dowling and Younger, 1983, supra; 1986, Virology 152:50-57] from the anti-viral effects of interferon. The primary effect of interferon-treatment on VSV replication is an inhibition of the translation of viral mRNA [Samuel (1988) Prog. Nucl. Acid Res. Mol. Biol. 35:27-72]. However, when coinfected with vaccinia, translation of VSV mRNA was rescued (Whitaker-Dowling and Younger, 1983, supra; 1986, supra). It has been suggested that the vaccinia virus inhibitor of the interferon-induced P.sub.1 /elF-2.alpha. kinase may partly contribute to the interferon resistance of vaccinia virus and to the rescue of VSV and EMCV from the antiviral effects of interferon.
To date, several dsRNA binding proteins have been identified, including the human and mouse interferon-induced, dsRNA-dependent protein kinases (PKR) [Meurs et al. (1990) Cell 62:379-390; Icely et al. (1991) J. Biol. Chem. 266:16073-16077, respectively]; a human transactivator response element/Rev response element binding protein (TAR/RRE) [Gatignol et al. (1991) Science 251:1597-1600]; the vaccinia virus p25 protein [Ahn et al. (1990) Mol. Cell Biol. 10:5433-5441]; Escherichia coli RNase III protein [March et al. (1985) Nucl. Acids Res. 13:4677-4685]; the Drosophila staufen gene product [St. Johnston et al. (1991) Cell 66:51-63]; the Saccharomyces pombe PacI protein [Ilino et al. (1991) EMBO J. 10:221-226]; Xenopus RNA-binding protein A [St. Johnston et al. (1993) Proc. Natl. Acad. Sci. 89:10979-10983]; the human son-a protein [St. Johnston et al. (1992) Proc. Natl. Acad. Sci. 89:10979-10983]; reovirus .sigma.3 protein [Imani et al. (1988) Proc. Natl. Acad. Sci. 85:7887-7891]; and the porcine group C rotavirus NSP3 and p8 proteins [Langland et al. (1994) J. Virol 68:3821-3829]. Genes for these dsRNA binding proteins share homology in a region at the C terminus [Chang et al. (1993) Virology 194:537-547; St. Johnston et al. (1992) Proc. Natl. Acad. Sci. 89:10979-10983] having the dsRNA-binding domain consensus sequence:
R/K-E-F-X-X-G/A-X-G-R/K-S-T-K-R-K/R-E/D-A-K-N/Q-A-A-A-K-L-V/V-A-L/V-D/E (SEQ ID NO: 1). PA1 R/K-E-F-X-X-G/A-X-G-R/K-S-T-K/R-K/R-E/D-A-K-N/Q-A-A-A-K-L/V-A-L/V-D/E (SEQ ID NO: 1).
Retroviruses are the causative agents for an increasing number of diseases of higher organisms including: AIDS, HIV, various leukemias, feline leukemia, murine leukemia, several avian leukemias, various sarcomas of mice, rats, monkeys, birds, and cats, and other lymphotrophic diseases of man, including Adult T-Cell leukemia. Acquired Immune Deficiency Syndrome (AIDS), the recently most noteworthy of these diseases, is caused by a retrovirus which has been called HTLV-III, LAV, RAV or most recently HIV [Coffin et al. (1986) Science, 232:697]. HIV is one of a group of retroviral diseases which attacks the T4 lymphocytes thereby destroying the body's immune system [Anderson, (1984) Science 226:401-409; Weiss (1985) In RNA Tumor Viruses-II, Vol. 2, Cold Spring Harbor Laboratory, pp. 405-485]. The disease is uniformly fatal and no cure has been developed which either kills the virus in situ or replaces the lost elements of the body's immune system. Some experimental drugs show limited effects in stopping the virus, but to date there is no proven therapy or cure for the AIDS patient. The high mutation rate resulting in a wide variation in antigenicity of various strains of the virus makes it unlikely that a traditional vaccine for the virus will be developed soon.
Retroviral diseases differ from many other viral diseases in that the infective agent, a retrovirus, eventually becomes integrated in the host cell's genome. The retrovirus inserts its genome into a host chromosome, such that its genetic material becomes part of the genetic makeup of the infected cell and is then replicated with the cell as the cell divides and multiplies. It is this characteristic which makes retroviruses especially persistent and immune to traditional anti-viral treatment. There is as yet no way to kill the retrovirus without killing the host cell. Thus, there is no proven cure, nor is there any proven effective vaccine or pharmacological agent against any retroviral disease.
Details of the life cycle and replication of retroviruses are discussed in Weiss et al., RNA Tumor Viruses, vols. 1 and 2 (Cold Springs Harbor Laboratory 1984), which is incorporated herein by reference in its entirety. FIG. 1 summarizes a model of a retrovirus life cycle. The life cycle of retroviruses is unique among viruses. The cycle begins when an infectious particle enters a host cell and releases two identical RNA molecules. These molecules are "reverse transcribed" by special viral enzymes to produce double-stranded DNA which circularizes and inserts into the host chromosome. The inserted DNA virus or "pro-virus" is structurally very similar to a normal host gene. It is transcribed to produce RNA, like any host gene. This RNA can then be processed in three ways: (a) it can be directly translated into certain viral proteins; (b) it can be processed and spliced, and then translated to produce other viral proteins; or (c) it can be packaged, along with various viral proteins to make a newly infectious particle. In the case of HIV, the infectious particles continuously "bud off" the infected cells and bind to uninfected cells, beginning the cycle over again.
The retroviral particle which is the infectious agent contains in its interior two single-stranded positive-sense viral RNA molecules each between 7,000 to 11,000 nucleotide bases in length. These viral RNAs combine with certain viral proteins to form a viral core, the core being surrounded by a membrane. Imbedded in the membrane are viral glycoproteins which can specifically bind the viral particles to the appropriate host cell system. The viral core is assembled within the host cell and exits from the host cell, taking some of the host's membrane with it. Hence the membrane of the viral particle is derived directly from the host cell. The particle travels to an uninfected host cell, and due to the glycoprotein on its exterior binds to the new host cell and the life cycle repeats. Once the virus enters the cell, it is disassembled, releasing the two identical viral RNA molecules. These molecules are each composed of a sequence having specific functional regions making up the virus' "genomic structure."
The genome of any retrovirus is divided into three regions: the 5' terminus, the 3' terminus and a central region containing genes coding for proteins (see FIG. 2). The 5' terminus is further divided into four functional regions: the terminal redundancy (R), a unique sequence (U5), the primer binding site (PB- or PBS) and an untranslated sequence (L). The L region may contain a splice donor site for subgenomic mRNA. The 3' terminus is further divided into three functional regions: the primer-binding site for positive strand DNA synthesis (PB+ or PBS), a unique sequence (U3) and another copy of the terminal redundancy (R). The U5, U3 and R regions are sometimes collectively referred to as the long Terminal Repeat (LTR) region. Components of the LTR region are involved in integration of the retroviral genome into the genome of its host. All retroviruses contain these highly conserved regions.
The production of DNA from the infectious RNA occurs by a complex process called reverse transcription. The viral reverse transcriptase enzyme first complexes with a specific tRNA molecule supplied by the host cell. For example, in the case of the AIDS-related virus, it is lysine tRNA which complexes with the reverse transcriptase. The 3' end of the tRNA molecule remains free to hybridize with the primer binding site (PBS) of the retroviral genome. This is a sequence within the virus, which is complementary to the 5' end of the tRNA. Once the virus enzyme tRNA complex has been formed, the enzyme can make a new DNA molecule, using the RNA virus as a template, and using the tRNA as a "primer." As the process proceeds, the RNA of the resulting RNA/DNA complex is degraded, leaving single-stranded DNA. While reverse transcription continues, second-strand DNA synthesis beings from the poly-purine site upstream of the U3 region and continues in the opposite direction from the first-strand DNA synthesis. The RNA primer molecule is consequently degraded. The DNA genomic structure differs from the RNA genomic structure in having a redundant U3 region added to the 5' end, and a redundant U5 region added to the 3' end. This genomic structure resembles a normal gene, with U3 being the promoter, with structural genes in the center, and a U5 tail.
The exact process of how the DNA virus inserts into host chromosomes is not known. It is known that the DNA virus first becomes a circle, and that this involves the short inverted repeat sequences at the ends of the virus. These inverted repeats may be involved in some form of DNA hybridization which brings the ends of the virus together, allowing circularization. Subsequently, insertion into the chromosome is generally assumed to be mediated by an enzyme which recognizes the splice site in the circle and directs insertion of a single copy of the virus into a random site within the host chromosome.
The transcription of viral DNA from the DNA pro-virus within a chromosome occurs in a manner similar to the transcription of any host gene. The U3 region functions as a polymerase II promoter and transcription begins at the beginning of the R region. The U3 promoter, like eukaryotic promoters, generally requires a transcriptional activator protein, which turns the promoter "on." Transcription proceeds through most of the pro-virus and is terminated at the end of the 3' R region. As a result, the transcript is a recreation of the smaller and infectious single-strand RNA genome. A poly-A tail is attached to the 3' end of this RNA and the 5' end is capped, making this molecule similar to normal host messenger RNA.
The RNA which is transcribed from DNA can be directly translated into protein, like any mRNA within the host. Some viral RNA is not translated into protein but rather is packaged into infectious viral particles. Such packaging involves the binding of certain viral proteins to specific sequences of the viral genome. For example, in the RSV viral system, it is part of the GAG sequence which is one of the parts of the genome which binds to and is recognized by such proteins and have been shown to be necessary for packaging of the RNA. The RNA which is packaged into viral particles does not appear to be reverse-transcription-competent until "maturation" of the particle, i.e., after it has existed away from the host cell.
All retroviruses, including HIV, once inserted into the host chromosome, must have their genes translated into viral proteins. If viral proteins are not abundant, the retrovirus cannot efficiently propagate to other cells and is not cytopathic to the infected host cell [Dayton et al. (1986) Cell 44:941-947; Fisher et al. (1986) Nature 320:367-371]. Such proteins are not produced without the proper functioning of certain viral regulatory proteins. One of the key DNA/RNA-binding regulatory proteins for the retrovirus HIV is the TAT protein [Keegan et al. (1986) Science 231:699-704]. The TAT protein is essential to protein translation of HIV, and possibly also involved in RNA transcription. It is apparent that the TAT protein recognizes and binds to the nucleic acid sequence corresponding to the 5' end of the R region. A second activator gene ART has also been shown to be important in HIV translation [Sodroski et al. (1986) Nature 321:412-417]. DNA/RNA binding of the previously described activator proteins is essential to HIV replication. Therefore, introducing genes into host cells, i.e., gene therapy for humans or germline transformation for animals, which will code for modified proteins of the retrovirus which compete or interfere with TAT or ART, will effectively block retrovirus replication.
Past research efforts have been predominantly confined to two traditional anti-retroviral approaches: immunological prevention and pharmacological therapy, neither of which appear to be very promising for control of retrovirus diseases. Also, chemical repression of virus diseases has not generally been effective in eradicating any persistent virus, and certainly would not be expected to eradicate a retrovirus. Anti-viral chemicals tend to slow the progress of a virus and to bolster native defense mechanisms, but chemical treatments must be continuously applied and typically have undesirable side effects.
For these reasons, it is doubtful that any retroviral disease can be cured by the traditional anti-viral approaches. An alternative approach to inhibiting retrovirus replication is genetic inhibition by introducing antisense constructs into host cells (gene therapy).
In the field of human medicine, altering the genotype of the host has not been a desirable method of fighting infectious disease. However, it is now believed that gene therapy is the direction now and for the relative future [Anderson (1984) Science 226:401-409]. There are many pathogens for which conventional defenses appear inadequate, and where the use of RNA replication inhibitors might be feasible. Many of the cells that are infected by retroviruses are derived from hematopoietic stem cells. If these stem cells can be altered by the incorporation of genes or other nucleic acid sequences which will synthesize RNA inhibitors that are antagonistic to virus propagation, an efficient method to both effectively prevent and to treat these retroviral diseases will be apparent. Further, if the expression of the RNA replication inhibitors can be regulated in the desired cells, it has application to other genetic diseases.
It would therefore be desirable to provide methods and compositions for expression inhibitors of RNA replication which are particularly effective at the dsRNA-like structure of the TAT site.